Antigenic linear peptide compound

ABSTRACT

The novel antigenic linear peptide compound of this invention comprises a sequence of ten amino acids namely, serine-leucine-asparagine-proline-alanine-isoleucine-glycine-threonine-aspartic acid-lysine, all of the amino acids being in their L-forms with the exception of glycine. The compound has utility in vaccines for reducing the fertility of mammals.

BACKGROUND AND PRIOR ART

Mammalian spermatozoa have been known to be antigenic for many years.More recently, it has been demonstrated that mammalian sperm contain anantigenic enzyme, which is known as the C₄ isozyme of lactatedehydrogenase (LDH-X, LDH-C₄). LDH-C₄ has been isolated in purecrystalline form from mouse testes. Goldberg (1972) J. Biol. Chem.247:2044-2048. The enzyme has a molecular weight of 140,000 and iscomposed of four identical C subunits. The amino acid sequence andthree-dimensional structure of LDH-C₄ has been studied and partiallydetermined by a number of investigators. See Musick et al (1976), J.Mol. Biol. 104:659-668; and Wheat et al (1977) Biochem. & Biophys. Res.Comm., 74, No. 3:1066-1077. Wheat et al determined the sequence of theessential thiol peptide from amino acid 159 to 171, and found that to benearly identical to essential thiol peptides from other vertebrate LDHisozymes.

In 1974, Dr. Erwin Goldberg reviewed the effects of immunization withLDH-X (LDH-C₄) on fertility, and advanced the possibility that "by usinga defined macromolecular constituent of sperm it becomes possible toelucidate its primary structure in terms of amino acid sequence, to mapspecifically the antigenic determinant(s) responsible for includinginfertility, and then to construct synthetic peptides containing thesedeterminants. Possessing the capability for synthesizing a molecule withsuch properties, makes the immunological approach to fertility controlfeasible." Karolinska Symposia on Research Methods in ReproductiveEndocrinology, 7th Symposia: Immunological Approaches to FertilityControl, Geneva, 1974 202-222. However, such synthetic antigenicpeptides remained a goal and not an achievement, although theirtheoretical desirability had been recognized. In 1979, Dr. ErwinGoldberg summarized the state of the art as follows:

"In conclusion, and on a practical basis immunotherapy for birth controlrequires more than effectiveness, specificity, reversibility, andabsence of systemic side reaction. Rather large amounts of the antigenmust be available in unequivocally pure form. This condition probablycannot be met by a natural product enzyme antigen from sperm or testes.Rather, contraceptive technology requires a synthesizable peptidefragment retaining antigenicity and provoking a response which impairsfertility. Completion of the structural analysis of LDH-C₄ should allowmapping of antigenic determinants and synthesis of such peptides for usein a new contraceptive technology." "Recent Advances in Reproduction andRegulation of Fertility," G. P. Talwar, editor, Elsevier/North HollandBiomedical Press (1979).

SUMMARY OF INVENTION

It has now been discovered that an antigenic peptide can be prepared bysynthesizing a linear sequence of ten amino acids comprising:serine-leucine-asparagine-proline-alanine-isoleucine-glycine-threonine-asparticacid-lysine. All of the amino acids used to prepare the foregoingpeptide are in their L-form with the exeption of glycine which has onlyone form. The N-terminal is serine and the C-terminal is lysine.Although not known with certainty, it is believed that the foregoingsequence of ten amino acids corresponds to amino acids 211 to 220, ofLDH-C₄. This is contrary to a recently published tentative sequence. SeeMusick et al (1979) J. Biol. Chem. 254, No. 16:7621-7623.

DESCRIPTION OF INVENTION

The present invention relates to a novel antigenic linear peptide havinga chain length of ten amino acids. The formula of this peptide can berepresented as follows:

    N-Ser-Leu-Asn-Pro-Ala-Ile-Gly-Thr-Asp-Lys-C

In the following formula, the letter "N" designates the N-terminal aminoacid, while the letter "C" designates the C-terminal amino acid. Ser,Leu, Asn, Pro, Ala, Ile, Thr, Asp, and Lys represent the L-amino acidforms, respectively, of cysteine, serine, leucine, asparagine, proline,alanine, isoleucine, threonine, aspartic acid, and lysine, and Glyrepresents glycine.

The peptide compound of the present invention can be synthesized fromits constituent amino acids. For example, the synthesis can be carriedout by the Merrifield solid phase method, as described in J.A.C.S.85:2149-2154 (1963). This solid phase method for synthesizing sequencesof amino acids is also described in Stewart and Young, Solid PhasePeptide Synthesis (W. H. Freeman and Co., San Francisco, 1969), pages1-4. In this procedure, the C-terminal amino acid, such as serine forthe compound of this invention, is attached to chloromethylatedpolystyrene-divinylbenzene copolymer beads. Each subsequent amino acid,with suitable protecting group, is then added sequentially to thegrowing chain. For example, as described in the Merrifield article, theprotective group may be a carbobenzoxy group. By the procedure ofcoupling, deprotection, and coupling of the next amino acid, the desiredamino acid sequence and chain length can be produced. As a final step,the protective group is removed from the N-terminal amino acid (viz.lysine and the C-terminal amino acid is cleaved from the resin, using asuitable reagent, such as trifluoroacetic acid and hydrogen bromide.Since this synthesis procedure is well known, it is not believed that itwill be necessary to further describe it herein. The peptide of thisinvention can be prepared by this synthesis procedure for use inreducing the fertility of mammals.

To utilize the antigenic peptide of this invention in the form of afertility reducing vaccine, the peptide is conjugated to a carriermolecule, which is preferably a protein which itself elicits anantigenic response and which can be safely administered. For example,the peptide can be coupled to tetanus toxoid for administration byintramuscular injection. For example, a mixture of 1μ Mole tetanustoxoid, 60μ Moles antigenic peptide, and 18 millimoles 1-ethyl-3-(3dimethyl aminopropyl) carbodiimide hydrochloride reacted in water (pH6)for 12 hours at room temperature and 24 hours at 4° gives a productcontaining 3.5 moles of peptide/mole tetanus toxoid. Excess reactantscan be removed by dialysis or gel filtration. See Pique et al,Immuno-chemistry, 15:55-60 (1978). Alternatively, the peptide may becoupled using bisdiazotized benzidine (Bassiri et al, Endocrinology,90:722 (1972) or glutaraldehyde. To facilitate coupling to a protein anadditional amino acid such as cysteine may be attached to the N-terminalserine. For example, the compound prepared in a form for coupling wouldbe: N-Cys-Ser-Leu-Asn-Pro-Ala-Ile-Gly-Thr-Asp-Lys-C.

For intramuscular injection, the coupled peptide may be suspended in asterile isotonic saline solution, or other conventional vehicle, and, ifdesired, an adjuvant may be included. A preferred use of such a vaccineis for administration to human females. Antibodies will be formed, whichwill appear in the oviduct fluids and thereby achieve a significantreduction in fertility. For this purpose, the amount to be administeredwill range from about 1 to 10 milligrams (mg) of the antigenic peptide.

I claim:
 1. The antigenic linear peptide compound having the formula: N-Ser-Leu-Asn-Pro-Ala-Ile-Gly-Thr-Asp-Lys-C wherein N-Ser is N-terminal serine and Lys-C is C-terminal lysine, and Leu, Asn, Pro, Ala, Ile, Thr, and Asp are the L-Amino acid forms, respectively of leucine, asparagine, proline, alanine, isoleucine, threonine, and aspartic acid. and Gly is glycine.
 2. The antigenic linear peptide compound having the formula: N-Cys-Ser-Leu-Asn-Pro-Ala-Ile-Gly-Thr-Asp-Lys-C wherein N-Cys is N-terminal cystine and Lys-C is C-terminal lysine, and Ser, Leu, Asn, Pro, Ala, Ile, Thr, and Asp are the L-amino acid forms, respectively, of serine, leucine, asparagine, proline, alanine, isoleucine, threonine, and aspartic acid, and Gly is glycine. 